This invention relates generally to the fields of molecular biology and molecular medicine and more specifically to the identification of novel polypeptides comprising a noncleavable form of a Fas ligand and having a capacity to activate a Fas receptor-mediated pathway involved in programmed cell death, and pharmaceutical compositions thereof, and therapeutic and prophylactic methods employing such polypeptides.
Programmed cell death, or apoptosis, is a physiological process that guarantees that homeostasis is maintained between cell proliferation and cell differentiation in nearly all self-renewing tissues of multicellular organisms. Apoptosis permits the elimination of cells that are no longer necessary, are produced in excess, have developed improperly, or have sustained genetic damage. A variety of cell types appear to undergo cell death through such an apoptotic mechanism.
In addition to preserving normal tissue homeostasis, apoptosis also occurs in response to a various external stimuli, including cytotoxic lymphokines, radiation, chemotherapeutic agents, growth factor deprivation, hyperthermia, hormone withdrawal, and infection by some viruses. See. e.g., Kerr et al., Cancer 73:2013-2026 (1994), published erratum, Cancer 73(12):3108 (1994). Accordingly, apoptosis is an inducible phenomenon that can be regulated by mechanisms of regulation that are similar to those involved in other metabolic pathways.
Dysregulation of apoptosis has also been observed and is implicated in the development of diseases resulting from inappropriate cell death or inhibition of cell death. For example, apoptotic dysregulation has been observed in some types of cancer cells which survive for longer periods than corresponding normal cells. The inhibition or failure of the apoptotic mechanism may permit such cells to undergo mutations leading to a transformed or cancerous state. See. e.g., Korsmeyer, Blood 80:879-886 (1992). Inhibition or failure of the apoptotic cell death mechanism may also contribute to diseases of the immune system by allowing persistence of self-reactive B and T lymphocyte cells, thereby promoting autoimmune disorders. See. e.g., Watanabe-Fukunaga et al., Nature 356:314-317 (1992). Apoptotic dysregulation has also been observed in neurodegenerative diseases in which neurons die prematurely.
Apoptosis is mediated, at least in part, by a cell surface receptor protein known as the Fas antigen receptor (xe2x80x9cFasxe2x80x9d) (also known as xe2x80x9cAPO-1 antigenxe2x80x9d or xe2x80x9cCD95xe2x80x9d). Fas is an approximately 45-kDa (kiloDalton) type I transmembrane protein belonging to the tumor necrosis factor (xe2x80x9cTNFxe2x80x9d)/nerve growth factor (xe2x80x9cNGFxe2x80x9d) receptor family of proteins. Tanaka et al., Nature Med. 2:317-322 (1996); Tanaka et al., EMBO J. 14:1129-1135 (1995). Fas is expressed in a variety of tissues, including the thymus, liver, lung, intestine, heart, and kidney, and in various human cell types, including, for example, lymphocytes, hepatocytes, activated B and T cells, and neutrophils, and carcinoma cells, such as breast, colon, prostate and pancreatic cancer cells. Tanaka et al., Nature Med. 2:317-322 (1996); Kayagaki et al., J. Exp. Med. 182:1777-1783 (1995). As a receptor protein, Fas has been found to transduce extracellular signals into a cell and, as a result, can mediate or trigger apoptosis. Itoh et al., Cell 66:233-243 (1991).
Because Fas is expressed on the cell surface, its mechanism of action is believed to be regulated by interacting with or binding to another cell surface protein. One such known protein is Fas ligand (xe2x80x9cFasLxe2x80x9d), a 40-kDa type II transmembrane protein of the TNF family. FasL has been observed to mediate and induce apoptosis by binding to Fas. Takahashi et al., Int""l Immunol. 6:1567-1574 (1994); Abbas, Cell 84:655-657 (1996). Human FasL is a polypeptide of 281 amino acids divided into three distinct domainsxe2x80x94an intracellular (i.e., cytoplasmic) domain, a transmembrane domain, and an extracellular domain. See, e.g., EP Patent Application, Publ. No. 0 675 200 A1 (published Oct. 4, 1995, issued to Mochida Pharmaceutical Co., Ltd.). FasL is predominantly expressed in activated T cells, but is also expressed in a number of other cell types, including Sertoli cells in the testis and the stroma cells of the retina. Tanaka et al., Nature Med. 2:317-322 (1996).
The interaction between FasL and Fas has been shown to be critical to the regulation of cell number in a large number of tissue and organ systems. Nagata, Adv. in Immunol. 57:129-144 (1994); Nagata et al., Science 267:1449-1456 (1995). The Fas/FasL system has been implicated, for example, in the pathogenesis of fulminant hepatitis, GVHHD, and AIDS. Kayagaki et al., J. Exp. Med. 182:1777-1783 (1995). Loss of function of the Fas/FasL system has also been observed to result in lymphoproliferative disorders and to accelerate autoimmune disorders in humans and mice. Takahashi et al., Cell 76:969-976 (1994).
Conversely, exaggeration of the Fas system appears to cause tissue damage. Tanaka et al., Nature Med. 2:317-322 (1996). Fas expression is upregulated in hepatocytes transformed by human hepatitis C virus. Lymphocytes transformed with human immune deficiency virus (xe2x80x9cHIVxe2x80x9d), human T cell leukemia (xe2x80x9cHTLVxe2x88x921xe2x80x9d) or Epstein-Barr virus (xe2x80x9cEBVxe2x80x9d) express a high level of Fas and appear to be sensitive to Fasmediated apoptosis. Tanaka et al., Nature Med. 2:317-322 (1996).
The Fas/FasL interaction has been particularly well studied in the immune system. The activation of T cells through the T cell receptor (xe2x80x9cTCRxe2x80x9d) upregulates both Fas and FasL on such cells. In circumstances of low to moderate TCR stimulation, T cells proliferate. Under conditions of repetitive or high levels of TCR stimulation, T cells are driven toward apoptosis. This phenomenon has been termed xe2x80x9cAntigen Induced Cell Deathxe2x80x9d (xe2x80x9cAICDxe2x80x9d). The importance of AICD in regulating the immune system has been demonstrated in the LPR mouse. Nagata et al., Immunol. Today 16:39-43 (1995). This mouse strain, which has a spontaneous disruption in the Fas gene from the insertion of an endogenous retroviral element, has been shown to be defective in AICD. With age, such mice develop large numbers of non-functional T lymphocytes in lymphoid and non-lymphoid tissues and develop a number of autoimmune syndromes.
The Fas/FasL system also appears to contribute to the phenomenon of immune privilege. It has been shown that in the testis and the anterior chamber of the eye, constitutive expression of FasL may serve to limit the immune response by eliminating Fas-bearing T cells and possibly other Fas-bearing inflammatory cell types. Bellgrau et al., Nature 377:630-632 (1995); Griffith et al., Science 270:1189-1192 (1995). The Fas system appears to maintain the immune privilege by preventing activated lymphocytes from infiltrating the testis or testis. In the case of the testis, it has been shown that FasL-bearing Sertoli cells can be transplanted across major histocompatibility complex (xe2x80x9cMHCxe2x80x9d) barriers, with constitutive FasL expression protecting against alloimmune mechanisms. Bellgrau et al., supra. This demonstration has inspired a number of investigators to study the possibility of artificially conferring immune privilege to transplanted organs via the induction of FasL expression. It has also been shown that syngeneic myoblasts expressing FasL can protect allogenic islets from alloimmune destruction. Lau et al., Science 273:109-112 (1996).
The mechanism of action by which FasL interacts with Fas and mediates apoptosis has been examined rather extensively. Recent studies have shown that membrane-bound FasL is converted to a soluble 26-kDa form of FasL (xe2x80x9csFasLxe2x80x9d) by action of a matrix metalloproteinase-like enzyme. Kayagaki et al., J. Exp. Med. 182:1777-1783 (1995); Tanaka et al., Nature Med. 2:317-322 (1996); Tanaka et al., EMBO J. 14:1129-1135 (1995). Soluble human FasL is believed to comprise an amino acid sequence of the extracellular domain of FasL which has been proteolytically cleaved from membrane-bound FasL and released into the surrounding body fluid or cell culture supernatant. See. e.g., EP Patent Application, Publ. No. 0 675 200 A1, supra. Soluble FasL is an active form of FasL; it has been shown to bind to Fas antigen receptor and thus to induce apoptosis. Tanaka et al., Nature Med. 2:317-322 (1996).
That apoptosis is mediated by a soluble form of FasL is not surprising given that FasL is homologous with TNF. It is well known that a secretory (soluble) form of TNF is released from the membrane-bound form of TNF by metalloprotease action at the cell surface of activated macrophages and T cells. Kriegler et al., Cell 53:45-53 (1988). Soluble TNF has been detected in the serum of patients with malignant tumor cells or with septic shock. Beutler and Cerami, Nature 320:584-588 (1986); Vassalli, Ann. Rev. Immunol. 10:411-452 (1992). Soluble TNF has been shown to be a biologically active form of TNF which can kill target cells bearing its receptor. Widespread release of soluble TNF into the circulatory system of an individual has been observed to precipitate systemic tissue damage. See. e.g., Perez et al., Cell 63:251-258 (1990). TNF is believed to kill its targets by either cell-to-cell contact through the transmembrane form of TNF or by local release of the TNF secretory component into the circulatory system of an individual. Id.
The proteolytic cleavage of FasL from the cell membrane to which it is bound appears to be quite efficient because inhibitors of matrix metalloproteases have been found to increase greatly the expression of membrane-bound FasL. These results suggest that the majority of translated FasL is cleaved. Indeed, FasL cannot be detected on some cell types by fluorescence activated cell sorting without also employing metalloprotease inhibitors. Kayagaki et al., J. Exp. Med. 182:1777-1783 (1995).
Because soluble FasL is released into and circulates widely throughout the circulatory system, it has the ability to bind to Fas-expressing cells throughout the body, resulting in nonspecific and widespread cell death. The potential toxicity of systemic Fas activation has been shown by the administration of agonistic anti-Fas antibody to mice. Such mice died within hours of antibody administration due to rapid liver failure resulting from fulminant hepatocyte death. Ogasawara et al., Nature 364:806-809 (1993), published erratum, Nature 365:568 (1993). Measurable levels of soluble FasL have also been reported in a number of human disease states and soluble FasL has been implicated as the causative agent for the idiopathic hepatitis and bone marrow suppression which is often seen in patients suffering from such disorders. Tanaka et al., Nature Med. 2:317-322 (1996). The potential toxicity of soluble FasL is also expected on numerous other tissues that express Fas, since soluble FasL is released from the cell membrane and can be transported all over the body. Mice expressing bcl-2 in hepatocytes, for example, have been shown to be protected from anti-Fas antibody mediated hepatocyte death. However, such mice were not found to be protected from death. Rodriguez et al., J. Exp. Med. 183:1031-1036 (1996).
It would be desirable to be able to modulate the expression of FasL and the interaction of FasL with Fas so as to be able to selectively modulate and induce apoptosis of specific targets, including particular tissues, organs, and cells. It would be especially desirable to be able to regulate the production of soluble FasL so as to prevent and/or minimize the deleterious effects of soluble FasL in the body, including the widespread and nonspecific cell, organ, and tissue death resulting from the circulation of soluble FasL throughout the body and its indiscriminate and damaging effects on a wide variety of cells, tissues, and organs. Such regulation would allow for the treatment and/or prevention of various symptoms resulting from diseases and disorders characterized by inadequate stimulation of the Fas receptor-mediated pathway or by inappropriate action or stimulation by soluble FasL on Fas receptors on cells, tissues, and organs throughout the patient""s body. As noted above, such regulation is made difficult by the fact that membrane-bound FasL is cleaved proteolytically to form soluble FasL which is released into local or systemic circulation, thereby causing nonspecific tissue and cell damage locally and systemically. Such regulation would also allow for the treatment and/or prevention of various symptoms resulting from diseases and disorders characterized by inadequate or inappropriate stimulation of the Fas receptor-mediated pathway. Thus, a need exists for a noncleavable form of FasL which can be used in artificially and selectively modulating apoptosis in various cells, tissues, and organs, and for reagents, compositions, and methods employing such noncleavable forms of FasL for use in therapeutic and prophylactic treatment of disorders responsive to the Fas-mediated pathway. This invention meets these and other needs.
In one embodiment of the invention, polypeptides having a capacity to activate a Fas receptor-mediated pathway are provided. Many of these polypeptides comprise amino acid sequences having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:1, but differing from SEQ ID NO:1 by at least one mutation within a Fas ligand (FasL) protease recognition region which comprises an amino acid sequence within SEQ ID NO:1. The FasL protease recognition region comprises an amino acid sequence within SEQ ID No:1. The mutation inhibits proteolytic cleavage of the polypeptide from a first cell membrane to which the polypeptide is bound relative to proteolytic cleavage of SEQ ID NO:1 from a second cell membrane to which SEQ ID NO:1 is bound. Many such polypeptides bind to the Fas receptor, and in many instances, the Fas receptor-mediated pathway is apoptosis. Some such polypeptides comprise a full-length Pas ligand, while some polypeptides comprise an allelic or species variant of SEQ ID NO:1.
In many such polypeptides, the mutation comprises deletion of at least one amino acid residue from an amino acid sequence of the FasL protease recognition region, substitution of at least one amino acid residue in an amino acid sequence of the FasL protease recognition region, insertion of at least one additional amino acid residue into an amino acid sequence of the FasL protease recognition region, or any combination thereof. In some polypeptides, the mutation comprises deletion of at least one amino acid residue between residue 118 and residue 155 of SEQ ID NO:1 or an allelic or species variation thereof.
In another aspect of the invention, polypeptides comprising a modified FasL protease recognition region and having a capacity to activate a Fas receptor-mediated pathway are provided. The proteolytic cleavage of these polypeptides from a first cell membrane to which the polypeptide is bound is reduced relative to the proteolytic cleavage of wild-type FasL from a second cell membrane to which the wild-type FasL is bound. Usually, the modified FasL protease recognition region of such polypeptides is formed by a wild-type FasL protease recognition region having at least one amino acid mutation, and the Fas receptor-mediated pathway is apoptosis.
In another aspect, the invention provides polypeptides having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:1 and a capacity to activate a Fas receptor-mediated pathway. Such amino acid sequence differs from the sequence of SEQ ID NO:1 by at least one mutation within a FasL protease recognition region within SEQ ID NO:1. The FasL protease recognition region comprises an amino acid sequence within SEQ ID NO:1. The mutation inhibits proteolytic cleavage of the polypeptide from a cell membrane to which it is bound relative to proteolytic cleavage of SEQ ID NO:1 from a cell membrane to which SEQ ID NO:1 is bound.
In another aspect of the invention, polypeptides comprising an extracellular domain of a FasL are provided. The extracellular domain has a capacity to activate a Fas receptor-mediated pathway. Furthermore, such polypeptides are linked to a cell membrane by a linker between the extracellular domain and the cell membrane, such that on cleavage of FasL with a protease, such as a metalloprotease, the domain remains linked to the cell membrane.
In yet another aspect of the invention, fusion proteins having at least two components are provided. The first component comprises a polypeptide comprising an amino acid sequence having at least 70%, 80%, 85%, or 90% sequence identity with the amino acid sequence of SEQ ID NO:1, said sequence differing from SEQ ID NO:1 by at least one mutation within a FasL protease recognition region within SEQ ID NO:1, whereby the mutation inhibits proteolytic cleavage of the polypeptide from a first cell membrane to which the polypeptide is bound relative to proteolytic cleavage of SEQ ID NO:1 from a second cell membrane to which SEQ ID NO:1 is bound. The second component comprises a heterologous protein linked to a membrane of a cell.
In another aspect of the invention, pharmaceutical compositions comprising a polypeptide of the invention, as described above, in a pharmaceutically acceptable carrier are provided.
The invention also provides compositions comprising a vector containing a nucleic acid sequence encoding a polypeptide of the invention, as described above, and a pharmaceutically acceptable carrier. Some such compositions further comprise lipids complexed with the vector.
In addition, the invention provides nucleic acid sequences encoding a polypeptide of the invention, as described above.
In another aspect, the invention provides isolated host cells containing a nucleic acid sequence encoding a polypeptide of the invention. Some host cells are mammalian cells. Furthermore, for some such host cells, the polypeptide resides on the surface of the cell. Compositions comprising such cells are also provided.
Also provided by the invention are pharmaceutical compositions comprising a pharmaceutically acceptable carrier and an isolated host cell containing a nucleic acid sequence encoding a polypeptide of the invention, as described above.
In another aspect, the invention provides vectors containing a nucleic acid sequence encoding a polypeptide of the invention, as described above.
In yet another aspect of the invention, methods of alleviating a symptom of a patient suffering from a disorder that is characterized by inadequate or inappropriate stimulation of a Fas receptor-mediated pathway in a tissue or an organ of the patient are provided. Such methods comprise delivering to the tissue or organ of the patient an effective amount of a cell containing a nucleic acid sequence encoding a polypeptide of the invention, as described above.
The invention also provides methods of alleviating a symptom of a patient suffering from a disorder that is characterized by inadequate or inappropriate stimulation of a Fas receptor-mediated pathway in a cell, a tissue, or an organ of the patient. Such methods comprise delivering to the cell, tissue, or organ of the patient an effective amount of a polypeptide of the invention that is linked to a pharmaceutically acceptable carrier having an affinity for the cell, tissue, or organ.
In another aspect of the invention, methods of modifying the phenotype of cells are provided. Such methods comprise contacting the cells with a pharmaceutical composition such that the phenotype of the cells is modified. The pharmaceutical composition comprises a vector containing a nucleic acid sequence encoding a polypeptide of the invention, as described above, and a pharmaceutically acceptable carrier. Usually, in such methods, the nucleic acid sequence integrates into the genome of the cells and is expressed, or replicates autonomously of the genome of the cells and is expressed. For some such methods, the cells are from a tissue or an organ of a patient suffering from a disorder characterized by inadequate or inappropriate stimulation or action of a Fas receptor-mediated pathway, and the method further comprises delivering such cells to the tissue or organ of the patient after modifying the phenotype of the cells.
The invention also provides isolated organs or tissues on which a polypeptide of the invention resides. Such polypeptide results from expression of a nucleic acid sequence encoding the polypeptide. The nucleic acid sequence is integrated into the genome of cells of the organ or tissue, or replicates autonomously of the genome of the cells, and is expressed.
Also provided are methods of alleviating a symptom of a patient suffering from a disorder that is characterized by inadequate or inappropriate stimulation or action of a Fas receptor-mediated pathway which comprise introducing into the patient an organ or tissue of the invention, as described above.
In another aspect of the invention, methods for the prophylactic or therapeutic treatment of intolerance to a graft in a patient are provided. Such methods comprise delivering to the graft of the patient an effective amount of a polypeptide of the invention.
Also included are methods for the prophylactic or therapeutic treatment of intolerance to a graft in a patient which comprise delivering to the graft of the patient an effective amount of a polypeptide of the invention that is linked to a pharmaceutically acceptable carrier. In some such methods, the carrier has an affinity for the graft.
Additionally, the invention provides methods of protecting an organ or tissue from autoimmune destruction in a patient in need of such treatment. Such methods comprise delivering to the organ or tissue of the patient an effective amount of a polypeptide of the invention. In such methods, the polypeptide has an affinity for the organ or tissue.
Also provided are methods for the prophylactic or therapeutic treatment of intolerance to a graft in a patient in need of such treatment. Such methods comprise delivering to the patient a graft on which a polypeptide of the invention, as described above, resides. Such polypeptide results from expression of a nucleic acid sequence encoding the polypeptide. The nucleic acid sequence is integrated into the genome of cells of the graft, or replicates autonomously of the genome of the cells, and is expressed.